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Isopsoralen Induces Differentiation of Prechondrogenic ATDC5 Cells via Activation of MAP Kinases and BMP-2 Signaling Pathways.

Li L, Eun JS, Nepal M, Ryu JH, Cho HK, Choi BY, Soh Y - Biomol Ther (Seoul) (2012)

Bottom Line: Isopsoralen significantly enhanced the protein expression of BMP-2 in a time-dependent manner.PD98059 and SB 203580, inhibitors of ERK and p38 MAPK, respectively, decreased the number of stained cells treated with isopsoralen.Taken together, these results suggest that isopsoralen mediates a chondromodulating effect by BMP-2 or MAPK signaling pathways, and is therefore a possible therapeutic agent for bone growth disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Dental Pharmacology, School of Dentistry and Institute of Oral Bioscience, Brain Korea 21 Project, Chonbuk National University, Jeonju 561-756 ; College of Pharmacy, Woosuk University, Wonju 565-701.

ABSTRACT
Endochondral bone formation is the process by which mesenchymal cells condense to become chondrocytes, which ultimately form new bone. The process of chondrogenic differentiation and hypertrophy is critical for bone formation and as such is regulated by many factors. In this study, we aimed to indentify novel factors that regulate chondrogenesis. We investigated the possible role of isopsoralen in induction of chondrogenic differentiation in clonal mouse chondrogenic ATDC5 cells. Isopsoralen treatment stimulated the accumulation of cartilage nodules in a dose-dependent manner. Further, ATDC5 cells treated with isopsoralen were stained more intensely with Alcian blue than control cells, suggesting that isopsoralen increases the synthesis of matrix proteoglycans. Similarly, isopsoralen markedly induced the activation of alkaline phosphatase activity compared with control cells. Isopsoralen enhanced the expressions of chondrogenic marker genes such as collagen II, collagen X, OCN, Smad4 and Sox9 in a time-dependent manner. Furthermore, isopsoralen induced the activation of extracellular signal-regulated kinase (ERK) and p38 MAP kinase, but not that of c-jun N-terminal kinase (JNK). Isopsoralen significantly enhanced the protein expression of BMP-2 in a time-dependent manner. PD98059 and SB 203580, inhibitors of ERK and p38 MAPK, respectively, decreased the number of stained cells treated with isopsoralen. Taken together, these results suggest that isopsoralen mediates a chondromodulating effect by BMP-2 or MAPK signaling pathways, and is therefore a possible therapeutic agent for bone growth disorders.

No MeSH data available.


Related in: MedlinePlus

Effects of Isopsoralen on alkaline phosphatase activity and BMP-2 in ATDC5 cells. ATDC5 cells were treated with 0.05μM Isopsoralen and 10 μg/ml insulin for 21days, then extracted for alkaline phosphatase activity assay as described in the “Materials and methods”. The cells were harvested and homogenized in lysis buffer (50 mM Tris pH7.4, 150 mM NaCl 1 mM EDTA), and 50 μg of the cell lysates was used to measure the alkaline phosphatase activity at 405 nm (A). The ATDC5 cells were treated with 0.05 μM Isopsoralen or 10 μg/ml insulin for 1, 2, 3 days and cells lysates were immunoblotted with an antibody against BMP-2 (B). The asterisk(*) indicates a significant difference (p<0.05) compared to the control. Each graph represents the mean ± S.E.M. (n=3).
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Figure 004: Effects of Isopsoralen on alkaline phosphatase activity and BMP-2 in ATDC5 cells. ATDC5 cells were treated with 0.05μM Isopsoralen and 10 μg/ml insulin for 21days, then extracted for alkaline phosphatase activity assay as described in the “Materials and methods”. The cells were harvested and homogenized in lysis buffer (50 mM Tris pH7.4, 150 mM NaCl 1 mM EDTA), and 50 μg of the cell lysates was used to measure the alkaline phosphatase activity at 405 nm (A). The ATDC5 cells were treated with 0.05 μM Isopsoralen or 10 μg/ml insulin for 1, 2, 3 days and cells lysates were immunoblotted with an antibody against BMP-2 (B). The asterisk(*) indicates a significant difference (p<0.05) compared to the control. Each graph represents the mean ± S.E.M. (n=3).

Mentions: The enzymatic activity of ALP was determined in cells treated with 0.05 μM isopsoralen and compared to that in insulin-treated cells. In our study, ALP activity was increased 1.5-fold in cells treated with 0.05 μM isopsoralen compared to control cells, while ALP activity also significantly increased 3.7-fold following treatment with 10 μg/ml insulin compared to control cells after 14 days (Fig. 4A). To determine whether isopsoralen


Isopsoralen Induces Differentiation of Prechondrogenic ATDC5 Cells via Activation of MAP Kinases and BMP-2 Signaling Pathways.

Li L, Eun JS, Nepal M, Ryu JH, Cho HK, Choi BY, Soh Y - Biomol Ther (Seoul) (2012)

Effects of Isopsoralen on alkaline phosphatase activity and BMP-2 in ATDC5 cells. ATDC5 cells were treated with 0.05μM Isopsoralen and 10 μg/ml insulin for 21days, then extracted for alkaline phosphatase activity assay as described in the “Materials and methods”. The cells were harvested and homogenized in lysis buffer (50 mM Tris pH7.4, 150 mM NaCl 1 mM EDTA), and 50 μg of the cell lysates was used to measure the alkaline phosphatase activity at 405 nm (A). The ATDC5 cells were treated with 0.05 μM Isopsoralen or 10 μg/ml insulin for 1, 2, 3 days and cells lysates were immunoblotted with an antibody against BMP-2 (B). The asterisk(*) indicates a significant difference (p<0.05) compared to the control. Each graph represents the mean ± S.E.M. (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3794527&req=5

Figure 004: Effects of Isopsoralen on alkaline phosphatase activity and BMP-2 in ATDC5 cells. ATDC5 cells were treated with 0.05μM Isopsoralen and 10 μg/ml insulin for 21days, then extracted for alkaline phosphatase activity assay as described in the “Materials and methods”. The cells were harvested and homogenized in lysis buffer (50 mM Tris pH7.4, 150 mM NaCl 1 mM EDTA), and 50 μg of the cell lysates was used to measure the alkaline phosphatase activity at 405 nm (A). The ATDC5 cells were treated with 0.05 μM Isopsoralen or 10 μg/ml insulin for 1, 2, 3 days and cells lysates were immunoblotted with an antibody against BMP-2 (B). The asterisk(*) indicates a significant difference (p<0.05) compared to the control. Each graph represents the mean ± S.E.M. (n=3).
Mentions: The enzymatic activity of ALP was determined in cells treated with 0.05 μM isopsoralen and compared to that in insulin-treated cells. In our study, ALP activity was increased 1.5-fold in cells treated with 0.05 μM isopsoralen compared to control cells, while ALP activity also significantly increased 3.7-fold following treatment with 10 μg/ml insulin compared to control cells after 14 days (Fig. 4A). To determine whether isopsoralen

Bottom Line: Isopsoralen significantly enhanced the protein expression of BMP-2 in a time-dependent manner.PD98059 and SB 203580, inhibitors of ERK and p38 MAPK, respectively, decreased the number of stained cells treated with isopsoralen.Taken together, these results suggest that isopsoralen mediates a chondromodulating effect by BMP-2 or MAPK signaling pathways, and is therefore a possible therapeutic agent for bone growth disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Dental Pharmacology, School of Dentistry and Institute of Oral Bioscience, Brain Korea 21 Project, Chonbuk National University, Jeonju 561-756 ; College of Pharmacy, Woosuk University, Wonju 565-701.

ABSTRACT
Endochondral bone formation is the process by which mesenchymal cells condense to become chondrocytes, which ultimately form new bone. The process of chondrogenic differentiation and hypertrophy is critical for bone formation and as such is regulated by many factors. In this study, we aimed to indentify novel factors that regulate chondrogenesis. We investigated the possible role of isopsoralen in induction of chondrogenic differentiation in clonal mouse chondrogenic ATDC5 cells. Isopsoralen treatment stimulated the accumulation of cartilage nodules in a dose-dependent manner. Further, ATDC5 cells treated with isopsoralen were stained more intensely with Alcian blue than control cells, suggesting that isopsoralen increases the synthesis of matrix proteoglycans. Similarly, isopsoralen markedly induced the activation of alkaline phosphatase activity compared with control cells. Isopsoralen enhanced the expressions of chondrogenic marker genes such as collagen II, collagen X, OCN, Smad4 and Sox9 in a time-dependent manner. Furthermore, isopsoralen induced the activation of extracellular signal-regulated kinase (ERK) and p38 MAP kinase, but not that of c-jun N-terminal kinase (JNK). Isopsoralen significantly enhanced the protein expression of BMP-2 in a time-dependent manner. PD98059 and SB 203580, inhibitors of ERK and p38 MAPK, respectively, decreased the number of stained cells treated with isopsoralen. Taken together, these results suggest that isopsoralen mediates a chondromodulating effect by BMP-2 or MAPK signaling pathways, and is therefore a possible therapeutic agent for bone growth disorders.

No MeSH data available.


Related in: MedlinePlus